Methods and reagents for preserving rna in cell and tissue samples

ABSTRACT

This specification relates to the field of molecular biology and provides novel methods and reagents for preserving and protecting the ribonucleic acid (RNA) content of samples from degradation prior to RNA isolation. This preservation may be accomplished without ultra-low temperature storage or disruption of the tissue.

This application is a continuation application of U.S. application Ser.No. 10/354,727 filed Jan. 30, 2003, which is a continuation of U.S.application Ser. No. 09/771,256 filed Jan. 26, 2001, now U.S. Pat. No.6,528,641 issued Mar. 4, 2003, which is a continuation of PCTApplication No. PCT/US99/17375 filed Jul. 30, 1999, which claimspriority to and is a continuation-in-part of U.S. patent applicationSer. No. 09/127,435 filed Jul. 31, 1998, now U.S. Pat. No. 6,204,375issued Mar. 20, 2001. The entire disclosure of each of theabove-referenced applications is incorporated herein by reference in itsentirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the field of molecular biology andprovides a novel method and reagent for preserving and protecting theribonucleic acid (RNA) content of tissue or cell samples fromdegradation prior to RNA isolation.

2. Description of Related Art

Obtaining high quality, intact RNA is the first and often the mostcritical step in performing many fundamental molecular biologyexperiments. Intact RNA is required for quantitative and qualitativeanalysis of RNA expression by Northern blot hybridization, nucleaseprotection assays, and RT-PCR.

There are many published reports which describe methods to isolateintact RNA from fresh (or quick frozen) cells or tissues. Most of thesetechniques utilize a rapid cell disruption step in which the tissue isdispersed in a powerful protein denaturation solution containing achaotropic agent (e.g., guanidinium or lithium salt). This rapiddisruption of cell membranes and inactivation of endogenous ribonucleaseis critical to prevent the RNA from being degraded.

To obtain high quality RNA it is necessary to minimize the activity ofRNase liberated during cell lysis and to prevent RNA degradation fromother sources. This is normally accomplished by using isolation methodsthat disrupt tissues and inactivate or inhibit RNases simultaneously.For specimens low in endogenous ribonuclease, isolation protocolscommonly use extraction buffers containing detergents to solubilizemembranes, and inhibitors of RNase such as placental ribonucleaseinhibitor or vanadyl-ribonucleoside complexes. RNA isolation from morechallenging samples, such as intact tissues or cells high in endogenousribonuclease, requires a more aggressive approach. In these cases, thetissue or cells are quickly homogenized in a powerful protein denaturant(usually guanidinium isothiocyanate), to irreversibly inactivatenucleases and solubilize cell membranes. If a tissue sample can not bepromptly homogenized, it must be rapidly frozen by immersion in liquidnitrogen, and stored at −80° C. Samples frozen in this manner must neverbe thawed prior to RNA isolation or the RNA will be rapidly degraded byRNase liberated during the cell lysis that occurs during freezing. Thetissue must be immersed in a pool of liquid nitrogen and ground to afine powder using mortar and pestle. Once powdered, the still-frozentissue is homogenized in RNA extraction buffer. In the laboratory, quickfreezing of samples in order to delay RNA extraction carries the penaltyof a substantial increase in hands-on processing time. Processingmultiple samples with liquid nitrogen and mortar and pestle is extremelylaborious.

Quick freezing is even less convenient outside of the laboratoryenvironment, but is still considered a necessity by those in the field.Scientists in the field collecting specimens for analysis do not haveaccess to a high-speed homogenizer. They are forced to carry a supply ofliquid nitrogen or dry ice large enough to store samples until they canbe transferred to an ultra-low temperature freezer. Similarly, RNAextracted from human biopsy samples is usually partly or mostly degradedbecause pathologists do not routinely flash freeze specimens to preserveRNA.

There have been attempts to isolate RNA from archival samples that havenot been prepared by the flash-freezing methodology. For example, Esseret al., 1995 claim the isolation of full length RNA from cells fixedwith 5% acetic acid, 95% ethanol, with RNase inhibitors. However, inthis paper, isolated cells in suspension were fixed in aceticacid/ethanol solution at −20° C. and then held at 4° C. for a relativelyshort time. Unfortunately, testing by the Inventor has shown that theEsser et al. 95% ethanol/5% acetic acid solution does not meet theperformance standards required by the present invention. RNA recoveredfrom both tissue samples and spleen cells in suspension kept at 4° C.for 20 hours appeared partially degraded, while RNA isolated fromtissues stored at ambient temperature was completely degraded.Experiments reported in Esser et al. show that the method results inloss of RNA, due to leakage from the cells caused by ethanol. Using thatmethod, 70% of the RNA is lost immediately upon fixation, and after 1hour, 80% of the RNA is gone. Further, in a test where tissue samplesand spleen cells were stored in the 95% ethanol/5% acetic acid solutionat 25° C. overnight, the RNA of both the cell and tissue samples wascompletely degraded. Data is shown in FIG. 1.

The use of high purity, intact RNA is fundamental for performing variousmolecular biological assays and experiments such as Northern blothybridization, nuclease protection assays, RT-PCR and medical diagnosis.The intrinsic instability of RNA and the presence of RNases in samplesmakes the isolation of intact RNA a difficult procedure. Further, theisolation and assay of RNA-containing samples is typically timeconsuming and tedious. The contamination of a molecular biologylaboratory with RNases due to human error can have catastrophic results.Thus, there is an ongoing need to develop improved techniques, to makeRNA isolation and assay methods more sensitive, more specific, faster,easier to use and less susceptible to human error and handling. It wouldtherefore be advantageous in many instances, for research facilities touse automated RNA preservation protocol. For example, the presentinvention, could be combined with rapid RNA assay techniques orintegrated nucleic acid diagnostic devices (U.S. Pat. No. 5,726,012,U.S. Pat. No. 5,922,591, incorporated by reference) for efficient,automated RNA preservation and analysis.

U.S. Pat. No. 5,256,571 reports a cell preservative solution comprisinga water-miscible alcohol in an amount sufficient to fix mammalian cells,an anti-clumping agent and a buffering agent. At least one paper,Dimulescu et al., reports the apparent use of this fixative to preservecervical cancer cells and cord blood lymphocytes prior to RNA isolation.

A large body of literature suggests that ethanol and acetonecombinations are the best known fixatives for future recovery of nucleicacids from archival tissue. Yet, in view of the studies of theinventors, such ethanol/acetone mixture does not provide all of thedesired characteristics of an RNA preservation medium. The mixtures donot protect RNA at ambient temperature, does not allow for thepreservation of RNA in solid, multi-cell samples, and are alsoflammable, which makes it intrinsically less attractive as a general usereagent.

Some peripherally related art exists that deals with aspects ofpreserving or recovering RNA from fixed or preserved tissue samples.These reports include numerous evaluations of the suitability ofhistological fixatives to maximize the signal obtained by in situhybridization to detect (not recover) RNA in tissue samples (for exampleU.S. Pat. Nos. 5,196,182 and 5,260,048). Other reports detail methods torecover fragmented RNA from fixed tissues for limited molecular analysisby PCR™ (Koopman et al., Foss et al., Stanta et al., Houze et al.). Torecover this fragmented RNA, samples are typically treated withproteinase K to degrade the structural components of the tissue, thenthe RNA is extracted with a guanidinium-based solution. The RNArecovered from fixed tissue is of extremely poor quality, averaging insize of about 200 bases (Stanta 1991). This is probably due to a numberof factors including the action of endogenous RNase and cross-linking ofthe RNA in the intracellular matrix during fixation. Since the RNA ismostly degraded, it can not be used for northern analysis or nucleaseprotection assays. It can be used in RT-PCR, but only for amplificationof very small fragments.

The use of ammonium sulfate to precipitate proteins out of solution isknown, but the use of ammonium sulfate to preserve RNA does not, to theInventor's knowledge, appear in the art. Two reports describe the use ofammonium sulfate to investigate the folding and activity of mammalianribonuclease A (Allewell et al. and Lin et al.). Allewell et al.investigated the effects of ammonium sulfate on the folding and activityof RNase A. At pH 5.5, the activity of ribonuclease A is suppressed toapproximately 10% of the untreated control level across a broad range ofammonium sulfate concentration. This suppression of activity wasexpected by the authors. It appears to be due to a salt-induceddenaturation of the protein. Unfortunately, even 10% RNase activitywould substantially degrade the RNA of a sample over time. Therefore,this inhibition is not sufficient to protect RNA in many applications.When the ammonium sulfate is at pH 7.0, the activity of RNase A issuppressed at low concentrations as expected, but unexpectedly rises to110% of the level of the untreated control at higher concentrations(3M). The authors theorize that the combination of the neutral pH andthe high salt concentration forces a refolding of the protein into analternate, highly active configuration. However, the Allewell et al.group were examining the activity of pure RNase A in solution, ratherthan in a cellular sample containing many RNases.

In view of the above, there is a need for methods and reagents thatallow one to preserve and recover high quality, intact RNA from tissuesamples stored at near ambient or ambient temperature.

SUMMARY OF THE INVENTION

The present invention relates to a novel method and reagents forpreserving the RNA in tissue fragments at temperatures above thefreezing point of the preservatives for extended periods of time,including days to months, prior to RNA isolation. There is no priorreport disclosing any reagent or method similar to that described inthis application. This breakthrough alleviates the necessity of eitherimmediately processing samples to extract RNA, or the restriction ofonly isolating tissue at sites which have a supply of liquid nitrogen ordry ice.

The present application relates to compositions of RNA preservationmedia and methods of preserving RNA comprising: (1) obtaining anRNA-containing sample; and (2) treating the sample with an RNApreservation medium that infiltrates the sample, and protects the RNAfrom nucleases. In a preferred embodiment, the RNA preservation mediumbrings about the precipitation the RNA in the sample along with cellularprotein in the sample. This co-precipitation of the RNA and cellularproteins is believed to render the RNA inaccessible to nucleases viaphysical means, while the action of the RNA preservation mediumsimultaneously inactivates or inhibits the action of the nucleases.

In some preferred embodiments, the RNA preservation medium comprises asalt that precipitates the RNA in the sample along with the cellularprotein. In more presently preferred embodiments, the salt is a sulfatesalt, for example, ammonium sulfate, ammonium bisulfate, cesium sulfate,cadmium sulfate, cesium iron (II) sulfate, chromium (III) sulfate,cobalt (II) sulfate, copper (II) sulfate, lithium sulfate, magnesiumsulfate, manganese sulfate, potassium sulfate, sodium sulfate, or zincsulfate. In presently preferred commercial embodiments, the salt isammonium sulfate.

In RNA preservation media comprising salt, the salt is typically presentin a concentration sufficient to precipitate the RNA in the sample alongwith the cellular protein. The salt is typically present in aconcentration between 20 g/100 ml and the saturating concentration ofthe salt. Specifically, salt concentrations of 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140,or 150 g/100 ml may be used, and the concentration may be a rangedefined between any two of these concentrations.

Of course, during use, some dilution of the salt concentration may occurdue to, for example, liquid in the sample. Therefore, these saltconcentrations may be higher than the final salt concentrations obtainedin some uses. Further, it is contemplated that amounts of salt above thesaturating concentration may be used in regard to the present invention.In such embodiments, there may be salt that is not in solution in theRNA preservation medium. This should not affect the RNA preservationabilities of the media. In fact, media having more than a saturatingconcentration of a salt may have some utility in applications where themedia is added to a liquid sample. In such cases, upon addition to theliquid sample, salt which is not in solution prior to addition, maybecome soluble due to the increase in liquid volume. Thus, the finalconcentration of a salt can be at a level higher than that possible if apreservation medium containing a saturating or less than saturating saltconcentration were used.

Preferred salts that have a solubility of greater than 20 g/100 ml are:ammonium sulfate, ammonium bisulfate, ammonium chloride, ammoniumacetate, cesium sulfate, cadmium sulfate, cesium iron (II) sulfate,chromium (III) sulfate, cobalt (II) sulfate, copper (II) sulfate,lithium chloride, lithium acetate, lithium sulfate, magnesium sulfate,magnesium chloride, manganese sulfate, manganese chloride, potassiumchloride, potassium sulfate, sodium chloride, sodium acetate, sodiumsulfate, zinc chloride, zinc acetate, or zinc sulfate.

In a preferred embodiment, the salt is ammonium chloride at aconcentration of between 20 g/100 ml and 100 g/100 ml, 30 g/100 ml and100 g/100 ml, or 30 g/100 ml and 80 g/100 ml. In a currently preferredcommercial embodiment, the salt is ammonium sulfate is in aconcentration of 70 g/100 ml.

The present invention is not limited to the use of ammonium sulfate, andother salts or compounds will also be useful in protecting RNA in tissuesamples and cell samples, for reasons as follows. The solubility ofindividual proteins depends greatly on the pH and salt concentration ofthe aqueous environment. Virtually all proteins are insoluble in purewater. As the ionic strength of the medium increases, proteins becomemore soluble. This is known as “salting in” of proteins. Above someionic strength, the solubility of protein decreases. The precisecondition at which this occurs is unique for each protein/saltcombination. In fact, at some salt concentrations, one protein may becompletely insoluble while another is at its solubility maximum. Thisphenomenon is known as “salting out.” Some salts have a much moredramatic salting out effect at high concentrations than others (e.g.,NO₃<Cl⁻<acetate⁻<SO₄ ²⁻). This phenomenon is the consequence of certaininherited characteristics of the ions (e.g., size, hydration, size,etc.). The present RNA protection media are believed to function due tothe salting out effect of high levels of salt. The theory is that thesalting out of proteins in the cells of the tissue samples or cellsamples is what leads to the formation of RNA-protective protein/RNAcomplexes. The importance of the “salting out” phenomenon to thisapplication is several-fold. First, it highlights that the effectivenessof RNA protection by protein precipitation using high concentrations ofsalt is complex and that certain combinations of ionic strength (saltconcentration) and pH may make a particular salt much more effective inone formulation than at a different pH or concentration. Second, itprovides a firm scientific foundation for the basic mechanism of actionby the reagents in this application, and guides one in the search foradditional RNA protective compounds within the scope of the invention.In order to determine whether another salt or putative RNA protectivecompound will function in the methods and reagents of the invention, oneneed merely obtain that salt or compound and test it in the mannersdescribed in the examples. By following the teachings of the examples,one of skill can easily elucidate whether a candidate substance isactually an RNA protective compound. Third, based on the theory thatprecipitation of intracellular proteins is the key to protecting RNA insitu, this explains why alcohol and acetone (agents that also canprecipitate protein, albeit by a different mechanism) are partiallyactive at protecting RNA in tissues, albeit not as protective as neededfor most applications.

In some embodiments, the RNA preservation medium will comprise acombination of at least two salts that precipitate the RNA in the samplealong with the cellular protein. In this manner, it might be that thetotal concentration of any given salt will not exceed 20 g/100 ml.However, in anticipated preferred embodiments, the combination of atleast two salts is present in a total salt concentration sufficient toprecipitate the RNA in the sample along with the cellular protein. Insome embodiments, the total salt concentration is between 20 g/100 mland 100 g/100 ml.

The RNA preservation medium may further comprise ethanol, methanol,acetone, trichloroacetic acid, 1-propanol, 2-propanol, polyethyleneglycol, or acetic acid. These additional potential components canprecipitate proteins in preserved cells and thereby protect RNA.However, these additional potential components are not salts. It isanticipated that in some embodiments, one will use these organicsolvents in combination with a concentration of salt to obtain one ofthe inventive RNA preservation media described herein. For example, itis anticipated that a combination of one or more of these organicsolvents in conjunction with less than 20 g/100 ml salt will accomplishthe goals of the invention.

In some embodiments, the RNA preservation medium comprises a salt suchas ammonium sulfate, ammonium bisulfate, ammonium chloride, ammoniumacetate, cesium sulfate, cadmium sulfate, cesium iron (II) sulfate,chromium (III) sulfate, cobalt (II) sulfate, copper (II) sulfate,lithium chloride, lithium acetate, lithium sulfate, magnesium sulfate,magnesium chloride, manganese sulfate, manganese chloride, potassiumchloride, potassium sulfate, sodium chloride, sodium acetate, sodiumsulfate, zinc chloride, zinc acetate, zinc sulfate, methanol,trichloroacetic acid, 1-propanol, 2-propanol, polyethylene glycol, oracetic acid. Further, the RNA preservation medium may comprise achelator of divalent cations, for example EDTA.

Typically, the RNA preservation medium comprises a buffer so that aconstant pH can be maintained. For example, the buffer can be sodiumcitrate, sodium acetate, potassium citrate, or potassium acetate. In apresently preferred commercial embodiment, the buffer is sodium acetate.Typically, the RNA preservation medium has a pH of between 4 and 8. Inpresently preferred commercial embodiments, the pH is 5.2.

The sample preserved in the RNA preservation media may be any of anumber of types of samples. For example, the sample may be a suspensionof cells, such as bone marrow aspirates, white blood cells, sperm,blood, serum, plasma, bacteria, tissue culture cells, or algae.Alternatively, the sample may a solid tissue, for example, the tissuesample is from brain, heart, liver, spleen, thymus, kidney, testis,ovary, tumors, tissue biopsies, plant stems, roots, or leaves. In somecases, the sample may comprise an entire organism. For example, theorganism may be a fish, insect, tadpole, coral, or embryo. In someprotocols, it will be of benefit to hold an organism or sample in theRNA preservation medium during dissection. For example, it might be ofbenefit to dissect an organism in the RNA preservation medium when thesample comprises an organism that is a pathogen within a tissue sampleor other organism. In this manner, the RNA of the pathogen maypreserved. Further, the RNA of the tissue sample or other organism ispreserved.

In many preferred methods, the practice of the invention will furthercomprise the step of isolating the preserved RNA. One of the advantagesof the present RNA preservation media is that the RNA may be isolatedfrom the tissue at a higher temperature than allowed with previoustechniques. For example, the RNA may be isolated at a temperature thatis greater than −20° C. In fact, the RNA may be isolated at roomtemperature.

In some cases, the sample may be stored in the RNA preservation mediumprior to the isolation of the RNA. For example, the tissue is storedunfrozen at −20° C. to 45° C. Owing to the salt content of some of theRNA preservation media, samples are not frozen at −20° C. In preferredembodiments the sample may be stored at greater than 0° C.

The invention also contemplates kits for preserving RNA within a sampleand isolating the RNA from the sample comprising: (1) an RNApreservation medium that infiltrates the sample and protects orpartitions the RNA from nucleases; and (2) a reagent for performing anRNA extraction from the sample. In some embodiments, the reagent forperforming an RNA extraction is a reagent for performing RNA extractionwithout organic solvents. Further, the reagent for performing an RNAextraction may be a reagent for performing a guanidinium-based RNAextraction. Alternatively, the reagent for performing an RNA extractionis a reagent for performing a lithium chloride-based RNA extraction.

In other embodiments of the present invention, a method of preservingRNA will comprise obtaining an RNA-containing sample, providing a saltand admixing the sample and the salt in a liquid to form an RNApreservation composition that infiltrates the sample and protects theRNA from nucleases. In one embodiment, the sample is comprised in theliquid prior to admixing the sample with the salt. In anotherembodiment, the salt is in a solid form prior to admixing with thesample and the liquid. In yet another embodiment, the salt is comprisedin the liquid prior to admixing the sample with the salt.

In one embodiment of the invention, the sample is a blood cell and theliquid is blood serum. In another embodiment the sample is urine. Inother embodiments, the liquid is water. In yet other embodiments, theliquid is a buffer.

In certain embodiments, RNA preservation compositions comprise obtainingan RNA-containing sample, providing a salt and admixing the sample andthe salt in a liquid. The liquid can be a component of the sample, oradded to the sample and salt. The salt is typically present in aconcentration sufficient to precipitate the RNA in the sample along withthe cellular protein. The salt is typically added so as to result in afinal concentration in solution between 20 g/100 ml and the saturatingconcentration of the salt. In some cases, it will be efficient to add avery concentrated salt, a saturated salt or a supersaturated salt to aliquid sample. Adding salt that is likely to result in a greater thansaturating concentration of salt in the final liquid sample has theadvantages of quickly allowing RNA preserving concentrations to bereached and avoiding the need to carefully consider the amount of saltneeded to reach a specific concentration in a given sample. Further, anysalt that does not go into solution, will not affect the RNA preservingproperties of the composition. Specifically, final salt concentrationsof 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95, 100, 110, 120, 130, 140, or 150 g/100 ml may be used, and theconcentration may be a range defined between any two of theseconcentrations. In some cases, a liquid containing more than asaturating concentration of salt may be employed, with the expectationthat additional volume of liquid in the sample will result indissolution of salt concentration.

In preferred embodiments, the salt is a sulfate salt, wherein the saltis ammonium sulfate. The ammonium sulfate is present in solution at afinal concentration of between 20 g/100 mL and the saturatingconcentration of the salt. In preferred embodiments, the salt is presentin solution at a final concentration of between 30 g/100 mL and 80 g/100mL. In other embodiments, the RNA preservation composition comprises atleast two salts, wherein the total salt concentration is present insolution at a final concentration of between 20 g/100 mL and 100 g/100mL. In another embodiment, the RNA preservation composition comprises achelator of divalent cations. In still other embodiments, the RNApreservation composition comprises a buffer, wherein said buffer has apH between 4 and 8.

The solid components of the present invention (e.g., salts, buffers, andshelters), can be prepared to yield the desired final componentconcentrations in solution when added to an aqueous sample. The solidcomponents can further be provided as powders, tablets, pills or othersuitable formulations that provide the desired properties of an RNApreservation composition. Solid components can be directly added to asample, added to a sample/liquid mixture, or present in a collectionvessel prior to collection of a sample or sample/liquid mixture. Theaddition of excipients and bulking agents such as mannitol, lactose,starch, cellulose, and the like, to provide desired solidcharacteristics (i.e., improved solubility, storage stability, particledispersion) are also considered in the formulation of powders, tabletsand pills. The solid components of the present invention can be addedprior to sample collection, after sample collection or any combinationthereof.

In one embodiment of the invention, pre-measured aliquots of a solid orliquid RNA preservation composition can be loaded into sample collectionvessels, and an appropriate volume of a RNA-containing sample added. Thecollection vessel would then be agitated, dissolving any solidcomponents of the RNA preservation composition, minimizing operatorexposure to an RNA sample. For example, a solid RNA preservationcomposition could be any of the salts previously described. Thus, inparticular embodiments of the invention, stabilizing RNA in a biologicalspecimen such as blood or urine as described above is contemplated.

In one example, a vial for collecting specimen such as urine or bloodcould be supplied with pre-measured aliquots of a RNA preservationcomposition. Immediately following collection of said specimen, the vialcould be agitated, admixing the RNA-containing specimen (i.e., sample)with the RNA preservation composition. A blood collecting vacuum vialcomprising an outwardly projecting needle is contemplated for us in thepresent invention (U.S. Pat. No. 5,090,420, specifically incorporatedherein by reference in its entirety) for rapid collection and mixing ofRNA-containing samples. In one embodiment, automated RNA preservation iscontemplated. Automated RNA preservation methods would be less tedious,faster, easier to use and less susceptible to human error and handling.

Also contemplated for use with pre-measured aliquots of a RNApreservation composition of the present invention are clinical specimencollection kits adapted for shipment by mail (U.S. Pat. No. 5,921,396,specifically incorporated herein by reference in its entirety).RNA-containing samples could be collected (e.g., away from a laboratoryfacility) and mixed with pre-measured aliquots of a RNA preservationcomposition provided in the vial, preserving RNA for shipment to asuitable RNA analysis site.

Alternatively, a RNA preservation composition can be pressed into atablet or pill and stored in bulk. Tablets would be a convenientcomposition for storage and could be added in the correct quantity to asample of any size in any container type. Thus, in other embodiments ofthe present invention, RNA preservation components in the form oftablets or pills are contemplated for field collection of samples fromsuch sources as water reservoirs, sewage plants or the dairy industry.In certain instances, RNA preservation media comprising predeterminedfinal salt concentrations or supersaturated salts could be supplied aspackets. The RNA preservation salt or supersaturated salt packets wouldbe especially useful in field studies, as there may be limited space orresources for analytical equipment. For example, packets could besupplied, as pre-measured and packaged as aliquots for a 1 mL, 5 mL, 10mL, sample. Of course, any size packet, to accommodate a variety of saltquantities could be provided as either an anhydrous powder, hydrouspowder, or a powder and liquid packaged individually or any combinationthereof. Thus, to preserve RNA in a sample, one would simply add thepacket contents to a sample and mix.

Certain advantages of using a solid component RNA preservationcomposition would be weight savings in storage and transport, spills ofsolid components are less likely and reduced volume savings (i.e.,preserving samples with dry reagents would minimize the final volume ofthe sample).

In other embodiments of the invention, RNA preservation compositionsfurther comprise isolating the preserved RNA, wherein the RNA isisolated at a temperature that is greater than −20° C. In otherembodiments, the sample is stored prior to the isolation of the RNA,wherein the sample is stored at temperatures greater than 0° C.

In particular embodiments, a composition of matter comprising anRNA-containing sample, a liquid, and a salt in a concentrationsufficient to protect the RNA from nucleases. In preferred embodiments,the salt is a sulfate salt, wherein the sulfate salt is ammoniumsulfate. In other embodiments, the composition comprises a combinationof at least two salts. In another embodiment, the composition comprisesa buffer, having a pH between 4 and 8. In yet another embodiment, thecomposition comprises a chelator of divalent cations.

The inventor's research indicates that 3M ammonium sulfate at pH 7.0 isquite effective at preserving intact RNA in intact tissue samples at allbut extreme temperatures (37° C.-42° C.). It is proposed that uponapplication to the sample, the ammonium sulfate diffuses into the tissueand cells and causes cellular proteins to precipitate (probably alongwith RNA which is intimately associated with many different proteins invivo) in protected complexes. In addition, RNase, which is localized incytoplasmic vesicles, may also be precipitated and rendered inaccessibleto cellular RNA.

It is believed that the mode of action of the claimed invention and themode of action of the Allewell et al. mixture are different. If the modeof action of the inventive solutions were the same as seen by Allewellet al. one would expect the RNA isolated from tissues stored in theinventive buffers to be degraded. Allewell et al. reports that at pH 5RNase is more active than normal. Thus, if RNAlater™ acted by the samemechanism, one would expect a boosting of the activity of RNase.Obviously, this would limit the ability of the RNA preservation media topreserve RNA. Based on the observation that such degradation does notoccur, the present invention functions by a different mechanism thanthat described by Allewell et al.

The term “RNAlater™” is a trademark of Ambion, Inc., for certaincommercial formulations of the RNA preservation media disclosed herein.In general, the term “RNAlater™” is employed to denote the formulationdisclosed in Example 2, which is composed of 25 mM Sodium Citrate, 10 mMEDTA, 70 g ammonium sulfate/100 ml solution, pH 5.2. This reagentfunctions by rapidly infiltrating cells with a high concentration ofammonium sulfate, causing a mass precipitation of cellular proteins.Importantly, cellular structure remains intact. The advantage of this isthat cells can be preserved and still identified histologically.

Following long-standing patent law, the words “a” and “an,” when used inconjunction with the word “comprising” in the claims or specification,denotes one or more.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1—Alcohol and acetone are unsuitable for preserving cellularsamples.

Panel A at 4° C. (lanes 1-5: ethanol, acetic ethanol, acetone, aceticacetone, RNAlater™). Panel B shows identical lanes at 37° C. RNAisolated from mouse liver stored overnight at 4° C. (lanes 1-5) or 37°C. (lanes 6-10). Lanes 1 and 6, storage in ethanol. Lanes 2 and 7,storage in acidified ethanol pH 4.0. Lanes 3 and 8, storage in acetone.Lanes 4 and 9, storage in acidified acetone pH 4.0. Lanes 5 and 10,storage in RNAlater™.

FIG. 2—RNA in fresh tissue samples is labile.

Fresh mouse liver (lanes 1 and 2), testis (lanes 3 and 4), and spleen(lanes 5 and 6) samples were placed either in RNAlater™ (lanes 2, 4, and6) (25 mM Sodium Citrate, 10 mM EDTA, 70 gm Ammonium Sulfate/100 mlsolution, pH 5.0) or in a 4 molar guanidinium isothiocyanate (GITC)based RNA extraction solution (lanes 1, 3, and 5) at 4° C. for 12 hours.RNA was extracted and analyzed by gel electrophoresis. Intact RNA isobserved only in lanes corresponding to tissues preserved in RNAlater™(lanes 2, 4, and 6).

FIG. 3—Defining the effective concentration range of Ammonium Sulfatethat protects RNA in tissue.

Fragments of freshly isolated mouse liver were placed in RNAlater™ withvarious amounts of Ammonium Sulfate. Samples contained 0, 10%, 20%, 30%,40%, 50%, or 70% Ammonium Sulfate (lanes 1-7 respectively). Afterincubation at 4° C. for 24 hours, RNA was extracted from the sample andanalyzed by denaturing agarose gel electrophoresis.

FIG. 4—Enhancing the potency of RNAlater™ at extreme temperatures byoptimizing both pH and Ammonium Sulfate concentrations.

The effects of pH and Ammonium Sulfate at extreme temperatures wereassessed. Fresh mouse liver was stored at room temperature or 37° C. for24 hours in 4 formulations of RNAlater™ solution. Lane 1: pH 7.0, 70g/100 ml Ammonium Sulfate, Lane 2: pH 5.0, 55 g/100 ml Ammonium Sulfate,Lane 3: The original RNAlater™ formulation (which contained 55 g/100 mlAmmonium Sulfate at pH 7.0), Lane 4: pH 5.0 and 70 g/100 ml AmmoniumSulfate concentration

FIG. 5—The specificity of Ammonium Sulfate on the effectiveness ofRNAlater™.

Panel A and B: Fresh liver samples were incubated in 5 buffers for 3days at 25° C. (panel A) and 37° C. (panel B), lane 1; RNAlater™ withammonium carbonate, lane 2; with ammonium chloride, lane 3; withpotassium sulfate, lane 4; with magnesium sulfate lane 5; AmmoniumSulfate. Panel C: Fresh liver incubated overnight at 4° C. in RNAlater™containing: lane 1, Cesium Chloride; lane 2, Cesium Sulfate; lane 3,Ammonium Sulfate.

FIG. 6—RNA isolated from mammalian tissues stored at 4° C. in RNAlater™.

Fresh mouse brain, heart, kidney, liver, and spleen (lanes 1-5) wereplaced in RNAlater™ solution and stored at 4° C. After incubation of oneweek (panel A), 2 weeks (panel B), and 4 weeks (panel C), RNA wasextracted from the tissue samples and analyzed by denaturing agarose gelelectrophoresis.

FIG. 7—RNA isolated from mammalian tissues stored at ambient temperature(25° C.) in RNAlater™.

Fresh mouse brain, heart, kidney, liver, and spleen (lanes 1-5) wereplaced in RNAlater™ solution and stored at 25° C. (ambient temperature).After incubation of two weeks (panel A) or 4 weeks (Panel B), RNA wasextracted from the tissue samples and analyzed by denaturing agarose gelelectrophoresis.

FIG. 8—RNA isolated from mammalian tissue stored at extreme temperatures(37° C.) in RNAlater™.

Fresh mouse liver, kidney, and spleen (lanes 1-3) were placed inRNAlater™ solution and stored at 37° C. After incubation for three days,RNA was extracted from the tissue samples and analyzed by denaturingagarose gel electrophoresis

FIG. 9—Using the Ambion to make RNA from tissues preserved in RNAlater™.

Lanes 1-3 are liver, heart, and kidney, ToTally RNA™, lanes 4-6 areRNAqueous™. Fresh mouse liver (lanes 1 and 4), heart (lanes 2 and 5),and kidney (lanes 3 and 6) samples were placed in RNAlater™ and storedovernight at 4° C. RNA was isolated from size matched samples usingeither the Ambion ToTally RNA™ kit (lanes 1-3) or the Ambion RNAqueous™kit (lanes 4-6). Lane 1—molecular weight marker.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention relates to the field of molecular biology andprovides novel methods and reagents for preserving and protecting theribonucleic acid (RNA) content of RNA-containing samples fromdegradation prior to RNA isolation. Strikingly, this is accomplishedwithout ultra-low temperature storage or disruption of the samples. Forexample, isolated human biopsy tissue can be placed in a RNApreservation medium and stored refrigerated for an extended period oftime, until the researcher has time to extract RNA for analysis.

The following examples illustrate the utility of the present invention.All examples make use of fresh animal or plant tissues, living cells insuspension or other samples containing RNA. The methods and compositionsare applicable for the preservation of RNA from a broad range ofbacterial, plant, and animal species, including humans. RNA samplesrecovered in these experiments were analyzed by formaldehyde/agarose gelelectrophoresis, staining with ethidium bromide, and illumination with300 nm ultraviolet light, as described in Molecular Cloning, ALaboratory Manual. (Maniatis, et al.).

The examples are included to demonstrate preferred embodiments of theinvention. It should be appreciated by those of skill in the art thatthe techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Criteria for Analysis of RNA to Determine if it is “Intact”

The Inventor routinely performs assays on RNA designed to assess theintactness of such samples. This criterion was used in the examples thatfollow to objectively gauge the quality of the RNA recovered fromtissues preserved in RNAlater™, other inventive RNA preservation media,or other solutions.

RNA is analyzed by electrophoresis on formaldehyde agarose gel using thebasic protocol described in “Molecular Cloning, a Laboratory Manual”(Maniatis, Fritsch, Sambrook, eds., Cold Spring Harbor Press). In orderto visualize the RNA in the gel, the intercalating dye Ethidium Bromideis added to the samples. Ethidium Bromide, when intercalated intonucleic acid, fluoresces under ultraviolet light, permittingvisualization of the nucleic acid. Intact RNA appears as a broad smearof heterogeneous mRNA (from 0.5 kilobase up to ˜10 kilobase), with twovery prominent, discrete bands, (28S and 18S ribosomal RNA),superimposed on the background smear in a 2:1 ratio. There should bevery little evidence of discrete bands intermediate in size between 18Sand 28S RNA. Partially degraded RNA is characterized by a loss of highmolecular heterogeneous RNA, multiple smaller ribosomal RNA cleavageproducts, and a deviation from the 2:1 ratio of 28S to 18S (28S is moresensitive to degradation). Severely degraded RNA will have 28S:18Sratios less than 1:1, and exhibit a smear of degraded ribosomal RNA from˜2 kilobase down to <0.1 kilobase.

“Partially degraded,” as used in this specification means that the ratioof 28S:18S rRNA is aberrant and may be as low as 1:1, but the rRNA bandsare still distinct.

“Mostly degraded,” as used in this specification means that the ratio of28S:18S is below 1:1. 28S may be barely visible, but there is stillnucleic acid in a smear extending from the approximate position of 28SrRNA down.

“Completely degraded,” and “degraded,” as used in this specificationmeans that the only RNA present is in a low molecular weight smear belowthe normal position of 18S rRNA

Example 2 Preparation of an Exemplary RNAlater™ RNA Preservation Medium

The description in this example provides one manner in which RNAlater™can be prepared. First, one should prepare or obtain the following stocksolutions and reagents: 0.5 M EDTA disodium, dihydrate (18.61 g/100 ml,pH to 8.0 with NaOH while stirring); 1M Sodium Citrate trisodium salt,dihydrate (29.4 g/100 ml, stir to dissolve); Ammonium Sulfate, powdered;Sterile water.

In a beaker, combine 40 ml 0.5 M EDTA, 25 ml 1M Sodium Citrate, 700 gmAmmonium Sulfate and 935 ml of sterile distilled water, stir on a hotplate stirrer on low heat until the Ammonium Sulfate is completelydissolved. Allow to cool, adjust the pH of the solution to pH 5.2 using1M H₂SO₄. Transfer to a screw top bottle and store either at roomtemperature or refrigerated.

Example 3 Exemplary General Manner of Preserving Tissue in RNAPreservation Media

Tissue samples to be stored in RNAlater™ or other inventive RNApreservation media should be excised from the source as quickly aspossible and placed in RNAlater™ or other inventive RNA preservationmedia. Some tissue samples may have a protective membrane or otherbarrier that would impede the rapid infusion of RNAlater™ or otherinventive RNA preservation media, such as a waxy coating on a leaf orthe capsule of a kidney or testis. These protective barriers should bedisrupted to allow rapid infiltration of the RNAlater™ or otherinventive RNA preservation media into the sample. In addition, largesamples should be dissected into smaller fragments to maximizediffusion. As a general guideline, sample thickness would be limited to0.5 cM in at least two dimensions. Samples that consist of cells insuspension should be concentrated into a small volume by gentlecentrifugation at a g-force sufficient to pellet the cells withoutdamage, resuspended in a minimal volume of the removed supernatant, thenmixed with 5 volumes of RNAlater™ or other inventive RNA preservationmedia (v/v). If concentration is not possible, the cell suspensionshould be diluted in 10 volumes of RNAlater™ or other inventive RNApreservation media. Alternatively, any other volume of liquid, essentialmedia or amount of a solid RNA preservation composition which results inthe preservation of RNA scan be used. Since the buffer will not disruptthe cells, concentration by centrifugation can be performed later.

Samples that are to be stored for 1 week or less may be stored atambient temperature (25° C.). For extended stability, samples should bestored refrigerated. For permanent archival storage (months-years),samples may be stored in a standard freezer (−20° C.). To isolate RNAfrom treated samples, tissue samples should be transferred directly intotissue extraction buffer. Cells in suspension must be pelleted bycentrifugation, then resuspended in RNA extraction buffer and processed.

Example 4 RNA in Fresh Tissue Samples is Labile

Fresh mouse liver, testis, and spleen samples (−0.5 cm³) were placedeither in RNAlater™ or in a 4 molar guanidinium isothiocyanate (GITC)based RNA extraction solution, or water at 4° C. for 12 hours. Thisguanidinium solution is a typical RNA extraction buffer (and found inAmbion's commercially available ToTally RNA™ and RNAqueous™ kits). GITCis a powerful chaotropic agent used either alone or in conjunction withother reagents in virtually all RNA isolation protocols. After anovernight incubation, RNA was extracted from the tissue samples andanalyzed by denaturing agarose gel electrophoresis. Intact RNA wasobserved only in lanes corresponding to tissues preserved in RNAlater™.RNA extracted from samples stored in GITC was badly degraded. Thus, GITCdoes not preserve the RNA in intact tissue samples. Animal tissuesstored in water or biological buffers such as normal saline yield nomeasurable RNA after an identical overnight incubation. Data is shown inFIG. 2.

Example 5 Determining the Effective Concentration of Ammonium Sulfatethat Protects RNA in Tissue

Freshly isolated mouse liver was placed in a variety of putative RNApreservation media with various concentrations of ammonium sulfate.Samples contained 0, 10%, 20%, 30%, 40%, 50%, or 70% Ammonium Sulfate.After incubation at 4° C. for 24 hours, RNA was extracted from thesample and analyzed by denaturing agarose gel electrophoresis.

In the sample with no ammonium sulfate, all that is observed is a lowmolecular weight smear. No specific banding of ribosomal RNA can beobserved. At 10% ammonium sulfate, slight evidence of rRNA bands can beobserved. At 20% ammonium sulfate, the 18S band is more obvious, andthere is more of a smear of degraded 28S rRNA extending down from theanticipated position of 28S rRNA. No specific 28S band is observed. At30% ammonium sulfate, both 28S and 18S rRNA bands are visible, howeverthere is extensive degradation (based on the aberrant ratio of 28S:18S,and numerous smaller bands observed. The sample with 40% ammoniumsulfate appears mostly intact, and the yield is 10 fold higher than at30%. While these experiments suggest a minimum requirement for 30-40%ammonium sulfate for protection of tissue RNA, the effectiveconcentration will depend on the type of tissue, the size of the tissuefragment, the storage temperature, and the period of storage.

Under more stringent incubation conditions, a higher concentration ofammonium sulfate is necessary for preservation of the RNA. For example,55 g/100 ml is required for protection of RNA in tissue samples at 25°C. and, as described in Example 6 70 g/100 ml appears required formaximal protection of RNA in tissue samples at 37° C. Further, ifsamples are stored in 55 g or less ammonium sulfate at 37 degrees for 24hours, at least some studies yield partially degraded RNA as judged by a1:1 ratio of 28S:18S rRNA. Data is shown in FIG. 3.

Example 6 Enhancing the Potency of RNA Preservation Media at ExtremeTemperatures by Optimizing both pH and Ammonium Sulfate Concentrations

The effect of pH and ammonium sulfate at extreme temperatures wasassessed. Fresh mouse liver was placed in 4 formulations of test RNApreservation media solution and stored at room temperature, or 37° C.,for 24 hours. RNA was extracted from the tissue samples and analyzed bydenaturing agarose gel electrophoresis. The formulation which contained55 g/100 ml ammonium sulfate at pH 7.0 was effective at ambienttemperature, but did not fully protect the RNA at 37° C. A combinationof low pH (5.0) and a higher ammonium sulfate concentration (70 g/100ml) was much more effective at high temperatures than the originalformulation and yielded intact RNA after 3 days at 37° C. Neithermodification alone (low pH or higher ammonium sulfate concentration)appreciably enhanced the stability of the RNA above that provided by theoriginal formulation. Data is shown in FIG. 4.

Example 7 The Specificity of Ammonium Sulfate on the Effectiveness ofRNA Preservation Media

Four additional formulations of test RNA preservation media wereproduced. The ammonium sulfate was replaced with saturating amounts ofammonium carbonate, ammonium chloride, potassium sulfate, or magnesiumsulfate. Fresh liver samples were incubated in these buffers for 3 daysat 25° C. and 37° C. RNA was extracted and analyzed by denaturingagarose gel electrophoresis. Only the ammonium sulfate control preventedthe RNA from being degraded. Maximum protection appears to require highconcentrations of both ammonium and sulfate ions. Ammonium sulfate isprobably the most effective of the salts tested because it is far moresoluble in water than these other salts, enabling formulations with veryhigh salt content. This hypothesis was challenged by evaluating twoesoteric (and more expensive) salts; cesium sulfate (which is quitesoluble—saturation at 362 g/100 ml) and cesium chloride (70 g/100 ml).These salts were substituted (in equal mass) for the ammonium sulfate inthe solution described in Example 2 and processed liver samples asabove. Cesium sulfate and cesium chloride offered partial protection tothe liver RNA at 4° C. Taken together, the data suggest that of thesalts tested, ammonium sulfate has superior ability to protect RNA fromdegradation in intact tissues. However, the partial protection theinventors see with high concentrations of other salt suggests a sharedmechanism of action, with varying degrees of efficacy. Data is shown inFIG. 5

Example 8 RNA Isolated from Mammalian Tissues Stored at 4° C. inRNAlater™

Fresh mouse brain, heart, kidney, liver, and spleen samples were placedin RNAlater™ solution, prepared as taught in Example 2, and stored at 4°C. After incubation of one week, 2 weeks, and 4 weeks, RNA was extractedfrom the tissue samples and analyzed by denaturing agarose gelelectrophoresis. All the RNA samples were intact. Fresh mouse brain,kidney, liver, and spleen samples were placed in 10 volumes of RNAlater™and stored at 4° C. After one week, two week, and four week incubations,equivalent weight fragments of tissue were removed and processed. Eachtissue sample was placed in a guanidinium isothiocyanate lysis solution,homogenized, and isolated using the Ambion RNAqueous™ kit (as describedin Example 20). The concentration of the RNA was determined by measuringthe absorbence at O.D.₂₆₀. The RNA was analyzed by formaldehyde-agarosegel electrophoresis as described in Example 1.

All RNA samples were judged “intact” based on clean, sharp ribosomal RNAbands in a 2:1 ratio of 28S:18S. In addition, overall quality was judgedby a visible background smear of heterogeneous RNA and a lack of visiblebreakdown products from ribosomal RNA. Data is shown in FIG. 6

Example 9 RNA Isolated from Mammalian Tissues Stored at AmbientTemperature (25° C.) in RNAlater™

Fresh mouse brain, heart, kidney, liver, and spleen samples were placedin RNAlater™ solution and stored at 25° C. (ambient temperature). Afterincubation for two or four weeks, RNA was extracted from the tissuesamples and analyzed by denaturing agarose gel electrophoresis asdescribed above. The RNA recovered after two weeks was intact. RNArecovered after a one month incubation was still ˜50% intact as judgedby the appearance of the 18S and 28S ribosomal RNA bands. Data is shownin FIG. 7

Example 10 RNA Isolated from Mammalian Tissue Stored at ExtremeTemperatures (37° C.) in RNAlater™

Fresh mouse liver, kidney, and spleen samples were placed in RNAlater™solution and stored at 37° C. After incubation for three days, RNA wasextracted from the tissue samples and analyzed by denaturing agarose gelelectrophoresis, as described above. The RNA isolated was intact. TheRNA isolated from tissues incubated one week-10 days is partiallydegraded (˜50% intact). This RNA is still a suitable substrate forNuclease Protection Assays or RT-PCR (Reverse Transcriptase-PolymeraseChain Reaction) both of which procedures are known to those of skill inthe art. Data is shown in FIG. 8.

Example 11 RNA Isolated from Amphibian, Fish, Insect, Bacteria, andPlant Tissue Stored at 4° C. in RNAlater™

The Inventor tested the effectiveness of RNAlater™ to preserve the RNAin Xenopus heart and liver, goldfish liver, whole beetle, wholeDrosophila, E. coli, tobacco, and alfalfa. Each were placed in RNAlater™solution and stored at 4° C. After incubation for 24 hours, RNA wasextracted from the samples and analyzed by denaturing agarose gelelectrophoresis as described in Example 1. All RNAs appeared intact.This demonstrates the effectiveness of RNAlater™ as a general reagentuseful for tissues from diverse organisms.

Example 12 Demonstration of the Suitability of RNAlater™ RNA as Targetin Northern Hybridization Analysis

Fresh mouse liver, kidney, and spleen samples were incubated inRNAlater™, for 24 hours at 25° C. or 37° C. RNA was extracted, resolvedby denaturing agarose gel electrophoresis, transferred to a positivelycharged nylon membrane (Brightstar Plus™ Ambion, Austin, Tex.), andhybridized with a radiolabeled antisense β Actin RNA probe according tomanufacturers recommendations (Northern Max™ Kit, Ambion, Austin, Tex.).The discrete signal corresponding to the 1.8 kilobase 13 Actintranscript detected in all samples indicate that the messenger RNA inall samples was completely intact and available for hybridization bynorthern analysis.

Example 13 The Suitability of RNAlater™ RNA as Template for RT-PCRAnalysis

RNA prepared from mouse liver, spleen, kidney, and testis stored inRNAlater™ for 24 hours at 37° C. was used as template for RT-PCR withtwo pairs of PCR™ primers for constitutively expressed genes (thehousekeeping genes cyclophilin and RIG/S 15) in a multiplex RT-PCRamplification. The expected products (216 bp cyclophilin, 324 bp RIG/S15) were obtained in all cases. Thus, the recovered RNA is suitable forRT-PCR analysis.

Example 14 Use of RNA Preservation Media to Protect Clinical Samples

There is an increasing trend towards genetic analysis of human clinicalsamples by RT-PCR. These clinical samples include solid tumors, isolatedcells, serum, urine, blood, or feces. Biopsies of solid tumors arefrequently analyzed for the expression of specific indicator genes likep53, whose aberrant expression plays a pivotal role in a number ofcancers. White blood cells isolated from normal or leukemic blood arefrequently analyzed for expression of interleukin genes. Urine, blood,serum, plasma, and feces are frequently analyzed for the presence ofpathogenic organisms. In an anticipated use, RNAlater™ or otherinventive RNA preservation media can be used as a holding buffer forpatient specimens isolated in a clinical setting. The RNA in thesesamples would then be protected until the samples could be transferredto a laboratory setting where intact RNA would be extracted foranalysis. In an anticipated use, RNAlater™ or other inventive RNApreservation media can be used to stabilize normally unstable viral RNA(such as HIV) in blood products for subsequent diagnosis.

Example 15 Use of RNA Preservation Media to Preserve Field Specimens

Field biologists throughout the world face the common problem of how topreserve samples collected in the field for later analysis in thelaboratory. Often research into RNA expression is simply avoided due tothe logistical difficulties involved in maintaining specimens frozen indry ice or liquid nitrogen. In an anticipated use, RNAlater™ or otherinventive RNA preservation media may be used as a holding buffer forspecimens isolated in the field by scientists who need to preservesamples until a later return to the laboratory environment. An importantbenefit of RNAlater™ or other inventive RNA preservation media is thatsmall specimens (such as microorganisms) remain intact. Thus, complexspecimens such as a population of microorganisms isolated from watersamples can be preserved en masse, sorted by classification in thelaboratory, then analyzed for gene expression.

Example 16

Use of RNA Preservation Media as a Dissection Medium

Biologists must frequently perform a complicated dissection in order toisolate a specimen for RNA analysis. Often the time delay means that RNAisolated from the sample will be of poor quality. Developmentalbiologists studying early mouse embryonic development must performmeticulous dissections to isolate early embryos from the decidua of theuterus. In another example, neuroanatomists must perform complicateddissections to remove a specific part of a brain for RNA analysis. In ananticipated use, RNAlater™ or other inventive RNA preservation mediawould be a very effective dissection medium. Immersing a sample inRNAlater™ or other inventive RNA preservation media will protect the RNAduring dissection, while preserving sample integrity (other reagents,such as guanidinium would cause extensive cellular lysis and compromisethe dissection). Using RNAlater™ or other inventive RNA preservationmedia would facilitate a lengthy dissection of a sample without the fearthat RNA in the sample will be degraded.

Example 17 Use of RNA Preservation Media to Preserve Samples forPathogenic Testing

Nucleic acid analysis to detect and classify pathogenic organisms is afast growing technology. In an anticipated use, RNAlater™ or otherinventive RNA preservation media may be used as a holding buffer forspecimens collected by health inspectors in the field. Placing samplesin RNAlater™ or other inventive RNA preservation media would preservethe RNA in the samples. The samples could then be analyzed using nucleicacid technology for the presence of pathogenic organisms. For example, aUSDA inspector could collect samples of meat from a slaughterhouse andplace them in RNAlater™ or other inventive RNA preservation media. TheRNA in pathogenic organisms present on the surface of the sample wouldbe preserved intact. The meat can later be removed from the sample, thepathogens recovered from the RNAlater™ or other inventive RNApreservation media, and the RNA isolated for analysis. One importantfeature of RNAlater™ or other inventive RNA preservation media is thatit is bactericidal, but does not lyse cells. Therefore, the titer ofpathogenic organisms will not change during storage of samples and skewapparent numbers, and samples can also be analyzed microscopically foradditional information.

Example 18 Use of RNA Preservation Media to Preserve Pathogens

Thousands of specimens each year are stored and shipped on dry ice tocentral laboratories that perform RNA analysis to detect pathogens. Inan anticipated use, RNAlater™ or other inventive RNA preservation mediamay be used as a shipping buffer, preserving RNA in infectious, diseasecausing organisms. RNAlater™ or other inventive RNA preservation mediawill permit samples to be shipped at ambient or near-ambienttemperatures without fear of RNA degradation. An organization that wouldbenefit both by the extra degree of protection RNAlater™ or otherinventive RNA preservation media would afford, plus realize asignificant savings in shipping costs is The Centers For Disease Control(CDC), who receive many human and animal samples for pathogenic testing.

Example 19 Use of RNA Preservation Media in FACS Sorting

Fluorescent Activated Cell Sorting (FACS) is a method with which cellsin suspension can be separated based on differences in cell surfacemarkers. We anticipate the use of RNAlater™ or other inventive RNApreservation media as a suspension solution for cells to be run througha FACS machine. In this way, the RNA in the cells would be preserved.Once the cells are sorted, intact RNA can be isolated for analysis.

Example 20 Use of RNA Preservation Media to Preserve Soil Bacteria

With the advent of RT-PCR methodology to identify bacterial species,there is a growing need for methods to isolate soil bacteria away fromsoil for subsequent RNA isolation. However, RNA in bacteria is extremelylabile and is rapidly degraded during the isolation protocol. Ananticipated use of RNAlater™ or other inventive RNA preservation mediais as a first step in RNA isolation from soil bacteria. Soil can bedispersed in RNAlater™ or other inventive RNA preservation media,instantly protecting the RNA within bacteria, but keeping the bacteriaintact. The soil can then be safely removed by low speed centrifugation,then the bacteria can be recovered by centrifugation at higher speed.The RNAlater™ or other inventive RNA preservation media will prevent theRNA from being degraded during the isolation procedure.

Example 21 Methods of Isolating RNA Preserved in RNAlater™ or OtherInventive RNA Preservation Media

Several methods of isolating RNA from tissue samples have been evaluatedfor suitability with RNA preservation media-preserved tissues. Severalsuch methods can be practiced with kits available from Ambion. Ofcourse, Ambion kits are not required for the isolation of RNA from RNApreservation media-preserved tissues, and the use of other methodologiesor kits to isolate RNA from RNA preservation media-preserved tissues andcell is covered by this specification. For example, any known methods ofisolating RNA, such as those of Boom et al. (selective binding andretention of nucleic acid to glass matrix—Qiaprep™, Qiagen, Inc.),Chomczynski et al. (Trizol™, MRC), Macfarlane et al. (Catrimox 14™, IowaBiotechnology, Inc.), Bugos et al. (Guanidine:Lithium Chloride), orAuffray et al. (LiCl:urea extraction), or kits for the practice of suchmethods may be used in this regard.

The following specific examples describe the use of Ambion kits toisolate RNA from tissue samples or cells stored in RNAlater™ or otherinventive RNA preservation media. It is contemplated that Ambion maychoose to sell a combined kit for the preservation of RNA in tissuesamples or cells, followed by the subsequent isolation of RNA from thosesamples.

Example 22 Isolation of Cellular RNA from Samples Preserved in RNAlater™or Other Inventive RNA Preservation Media Using the Ambion ToTally RNA™Kit

The Ambion ToTally™ RNA kit is a Guanidinium/Acid Phenol method ofpreparing cellular RNA.

In order to demonstrate the utility of combining RNAlater™ or otherinventive RNA preservation media preservation of tissue sample with theToTally RNA™ procedure, tissue samples were removed from storage inRNAlater™ and homogenized in 10 volumes of a Guanidinium Isothiocyanatelysis solution consisting of 4M guanidinium hydrochloride, 0.5%sarcosine, 25 mM Sodium Citrate, 0.1M 2-mercaptoethanol. Proteins areremoved by an extraction with an equal volume of phenol: chloroform(1:1), followed by a centrifugation to separate the aqueous and organicphases. The aqueous phase is recovered and extracted a second time withphenol at pH 4.7. This second extraction partitions any remainingproteins in the organic phase and the low pH forces any DNA into theorganic phase. The RNA remains in the aqueous phase.

The aqueous phase is recovered by centrifugation. The RNA is recoveredfrom the aqueous phase by precipitation with 0.3M Sodium Acetate and 2.5volumes of ethanol. Results are shown in FIG. 9.

Example 23 Isolation of Cellular RNA from Samples Preserved in RNAlater™or Other Inventive RNA Preservation Media Using the Ambion RNAqueous™Kit

The Ambion RNAqueous™ kit is a Guanidinium lysis method for isolatingRNA that does not require organic solvents. Instead, it relies upon theselective adsorption of RNA upon a glass fiber filter.

In order to demonstrate the utility of combining RNAqueous™ withRNAlater™ or other inventive RNA preservation media tissue samples wereremoved from storage in RNAlater™, transferred directly to theGuanidinium Isothiocyanate lysis solution provided in RNAqueous™ andhomogenized. The homogenate was applied to a glass fiber filter andwashed with several buffers, which remove protein and DNA. Pure RNA wasthen eluted from the filter with water. The coupling of RNAlater™ orother inventive RNA preservation media and RNAqueous™ has the advantagethat no caustic or carcinogenic organic reagents (phenol, chloroform,ethanol, acetic acid, etc) are used throughout the procedure. Freshsamples or samples preserved in RNAlater™ were homogenized in 10 volumesof a solution consisting of 4M guanidinium hydrochloride, 1% sarcosine,25 mM Sodium Citrate, 0.1M 2-mercaptoethanol, 2% Triton X-100. Thehomogenate is diluted 2× and passed over a glass fiber filter. Underthese conditions, nucleic acids bind to the filter and proteins arewashed away. Several sequential washes in high salt and ethanoldifferentially wash away the DNA, leaving only RNA bound to the filter.The RNA is subsequently recovered by elution with hot water. Results areshown in FIG. 9.

Example 24 Use of solid RNAlater to Preserve Nucleic Acid in LiquidSamples

The solid components of the preferred RNA preservation composition maybe dissolved directly in a liquid sample to be stabilized, added to asample/liquid mixture, added to a sample that has been placed in aliquid or present in a collection vessel prior to collection of a sampleor sample/liquid mixture.

The solid components can be blended in a dry mixer to the propercomposition such that when added to an aqueous sample and dissolved, thepreferred salt, buffer, and chelator concentration is achieved insolution. The pre-measured aliquots of powdered, dry components can beloaded into sample collection vessels, and an appropriate volume ofsample would be added. The collection vessel would then be agitated,dissolving the RNAlater components in the solution. This would minimizeoperator exposure to the sample, and would be a preferred method forstabilizing nucleic acids in a biological specimen such as blood orurine. Alternatively, the dry components can be pressed into tablet formand stored in bulk. Tablets would be a convenient format for storage andcan be added in the correct quantity to a sample of any size in any typeof container. This would be a preferred method for field collection ofliquid samples from such sources as water reservoirs or sewage plants orthe dairy industry. The primary advantages of using the RNA preservationreagent in dry mode would be weight savings in storage and transport,spills of solid components would be less likely, and volume savings inthat preserving liquid samples with dry reagents would minimize thefinal volume of the sample.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecomposition and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

-   U.S. Pat. No. 5,922,591-   U.S. Pat. No. 5,921,396-   U.S. Pat. No. 5,726,012-   U.S. Pat. No. 5,090,420-   Allewell, N. M., Sama, A., “The effect of ammonium sulfate on the    activity of ribonuclease A.” Biochemica et Biophysica Acta    341:484-488 (1974).-   Auffray, C., Rougeon, F., “Purification of mouse immunoglobulin    heavy chain messenger RNAs from total myeloma tumor RNA.” Eur. J.    Biochemistry June; 107(2)303-314 (1980).-   Boom, W. R., Adriaanse, H., Kievits, T., Lens, P. F., U.S. Pat. No.    5,234,809 entitled: Process for Isolating Nucleic Acid.-   Bugos R. C., Chiang, V. L., Zhang, X. H., Campbell, E. R., Podila G.    K., Campbell W. H., “RNA isolation from plant tissues recalcitrant    to extraction by guanidine.” Biotechniques November; 19(5)734-7    (1995).-   Cairns, M. T., Church, S., Johnston, P. G., Phenix, K. V.,    Marley, J. J., “Paraffin-embedded tissues as a source of RNA for    gene expression analysis in oral malignancy.” Oral Diseases    September; 3(3):157-161 (1997).-   Chomczynski, P., U.S. Pat. No. 5,346,994 entitled: Shelf Stable    Product and Process for Isolating RNA, DNA, and Proteins.-   Dimulescu et al., “Characterization of RNA in Cytologic Samples    Preserved in a Methanol-Based Collection Solution,” Molecular    Diagnosis 3(2):67-72 (1998).-   Esser, et al., “Isolation of full-sized mRNA from ethanol-fixed    cells after cellular immunofluorescence staining and    fluorescence-activated cell sorting.” (FACS). Cytometry 1995 Dec. 1;    21(4):382-386.-   Foss, R. D., Guha-Thakurta, N., Conran, R. M., Gutman, P., “Effects    of fixative and fixation time on the extraction and polymerase chain    reaction amplification of RNA from paraffin-embedded tissue.”    Comparison of two housekeeping mRNA controls. Diagn Mol Pathol    September; 3(3):148-155 (1994).-   Houze, T. A., Gustaysson, B., “Sonification as a means of enhancing    the detection of gene expression levels from formalin-fixed,    paraffin-embedded biopsies.” Biotechniques December; 21(6):1074-1078    (1996).-   Hurley et al., U.S. Pat. No. 5,256,571 entitled Cell Preservation    Solution (1993)-   Koopmans, M., Monroe S. S., Coffield, L. M., and Zaki, S. R.,    “Optimization of extraction and PCR amplification of RNA extracts    from paraffin-embedded tissue in different fixatives.” J. Virol    Methods July; 43(2):189-204 (1993).-   Lin L.-N., Brandts, J. F., “Refolding of ribonuclease in the    presence and absence of ammonium sulfate pulses.” Comparison between    experiments and simulations. Biochemistry 26:1826-1830 (1987)-   Macfarlane, D. E., U.S. Pat. No. 5,010,183 entitled: Process for    Purifying DNA and RNA using Cationic Detergents.-   Stanta G., Schneider, C., “RNA extracted from paraffin-embedded    human tissues is amendable to analysis by PCR amplification.”    Biotechniques September; 11(3):304 (1991).

1-10. (canceled)
 11. A method of preserving RNA comprising: (i)obtaining an RNA-containing sample; and (ii) treating the sample with anRNA preservation medium that infiltrates the sample and protects the RNAfrom nucleases.
 12. The method of claim 11, wherein the RNA preservationmedium precipitates the RNA in the sample along with cellular protein inthe sample and renders the RNA inaccessible to nucleases.
 13. The methodof claim 11, wherein the RNA preservation medium comprises a salt. 14.The method of claim 13, wherein the salt is a sulfate salt.
 15. Themethod of claim 14, wherein the salt is ammonium sulfate.
 16. The methodof claim 13, wherein the salt is present in a concentration between 20g/100 ml and the saturating concentration of the salt.
 17. The method ofclaim 13, wherein the salt is ammonium sulfate in a concentration ofbetween 30 g/100 ml and 80 g/100 ml.
 18. The method of claim 11, whereinthe RNA preservation medium comprises a combination of at least twosalts.
 19. The method of claim 18, wherein the total salt concentrationis between 20 g/100 ml and 100 g/100 ml.
 20. A kit for preserving RNAwithin a sample and isolating the RNA from the sample comprising: (i) anRNA preservation medium that infiltrates the sample and protects the RNAfrom nucleases; and (ii) a reagent for performing an RNA extraction fromthe sample.
 21. The kit of claim 20, wherein the reagent for performingan RNA extraction is a reagent for performing a guanidinium-based RNAextraction.
 22. The kit of claim 20, wherein the reagent for performingan RNA extraction is a reagent for performing a lithium chloride-basedRNA extraction.
 23. A method of preserving RNA comprising: (i) obtainingan RNA-containing sample; (ii) providing a salt; and (iii) admixing thesample and the salt in a liquid to form an RNA preservation compositionthat infiltrates the sample and protects the RNA from nucleases.
 24. Themethod of claim 23, wherein the sample is comprised in the liquid priorto admixing the sample with the salt.
 25. The method of claim 23,wherein the sample is a blood cell and the liquid is blood serum. 26.The method of claim 23, wherein the liquid is water.
 27. The method ofclaim 23, wherein the liquid is a buffer.
 28. The method of claim 23,wherein the salt is in a solid form prior to admixing with the sampleand the liquid.
 29. The method of claim 23, wherein the salt iscomprised in the liquid prior to admixing the sample with the salt. 30.The method of claim 23, wherein the salt is a sulfate salt.